Apparatus and method for separating and concentrating fluids containing multiple components

ABSTRACT

An apparatus that allows for separating and collecting a fraction of a sample. The apparatus, when used with a centrifuge, allows for the creation of at least three fractions in the apparatus. It also provides for a new method of extracting the buffy coat phase from a whole blood sample. A buoy system that may include a first buoy portion and a second buoy member operably interconnected may be used to form at least three fractions from a sample during a substantially single centrifugation process. Therefore, the separation of various fractions may be substantially quick and efficient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent application Ser. No. 10/932,882, filed on Sep. 2, 2004, entitled “APPARATUS AND METHOD FOR SEPARATING AND CONCENTRATING FLUIDS CONTAINING MULTIPLE COMPONENTS”, which is a continuation-in-part of U.S. patent application Ser. No. 10/445,381, filed on May 23, 2003, entitled “APPARATUS AND METHOD FOR SEPARATING AND CONCENTRATING FLUIDS CONTAINING MULTIPLE COMPONENTS” that claimed the benefit of U.S. Provisional Application No. 60/383,013, filed on May 24, 2002. The disclosures of the above applications are incorporated herein by reference.

FIELD

The present teachings relate to a multiple component fluid and a concentrator/separator and, more particularly, relates to a container operable with a centrifuge to separate and/or concentrate various biological components.

BACKGROUND

Various fluids, such as whole blood or various other biological fluids, may be separated into their constituent parts, also referred to as fractions or phases. For example, whole blood samples may include a plurality of constituents that may be separated by density in a device such as a centrifuge. The whole blood sample may be placed in a test tube, or other similar device, which is then spun in a centrifuge. In the centrifuge the whole blood is separated into different fractions depending upon the density of that fraction. The centrifugal force separates the blood or other sample into different fractions. In addition, various elements may be added to the test tube to create more than two fractions. In particular, commonly used gels may be used to divide the whole blood into a plurality of different fractions which may include fractions such as platelets, red blood cells, and plasma. Various other biological fluids may be separated as well. For example, nucleated cells may be separated and extracted from bone marrow or adipose tissue sample.

Many of these systems, however, do not provide a simple or efficient method to extract any more than one fraction and, especially, a fraction other than the top fraction. The top fraction of whole blood is plasma, or other blood constituents suspended in plasma. Thus, to extract other fractions the plasma fraction must either be removed for further extracting procedures or spun again to obtain the constituents suspended in this plasma. It is difficult to pierce the top fraction without commingling the sample. Accordingly, obtaining the other fractions is difficult with commonly known systems.

Other systems have attempted to alleviate this problem by providing a float or other device that is disposed within the sample at the interfaces of the different fractions during the centrifuge process. Nevertheless, these systems still do not allow a simple way to remove the different fractions without remixing the sample fractions. In addition, many of the systems do not allow an easy and reproducible method to remove the desired sample fraction.

Therefore, it is desired to provide a device to allow for the easy and reproducible removal of a particular fraction which does not happen to be the top fraction of a sample. It is desired to remove the required sample without mixing the different fractions during the extraction process. In addition, it is desired to provide a device which allows for a consistent extraction which includes known volumes or concentration of the fraction elements. Moreover, it is desired to separate and concentrate a selected fraction with one centrifugation step.

SUMMARY

The present teachings provide an apparatus that separates and concentrates a selected fraction or component of a fluid, such as a biological fluid. For example, undifferentiated cells, such as mesenchymal stem cells, platelet fraction, buffy coat, or white blood cell fraction can be separated from bone reaming material, whole blood, bone marrow aspirate, and other materials. In various embodiments, the apparatus, when used with a centrifuge, is generally able to create at least two fractions. The present teachings also provide for a new method of creating at least three fractions extracting a third fraction from a sample such as, for example, a buffy coat fraction.

In various embodiments, the apparatus includes a container to be placed in a centrifuge after being filled with a sample and the container includes a buoy or fraction separator having a selected density that may be less than one fraction but greater than a second fraction, that is disposed therein. In various embodiments, a second buoy may be placed in the container with the first. The extraction system is connected to the buoy system or to the collection chamber such that the fraction in the container can be collected and drawn outside of the chamber. During the centrifuge processing, the buoy is forced away from a bottom of the container as the denser fraction collects at the bottom of the container. The buoy is generally able to physically separate the denser fraction from another fraction of the sample. In various embodiments, the fractions can be withdrawn using an extraction system.

According to various embodiments, in addition to providing a first buoy and/or a second buoy, a buoy system may be provided. Generally, the buoy system may separate the sample into at least three fractions. The fractions may be separated and extracted from the container without substantially commingling the various fractions. Generally, a first buoy and a second buoy operate together to separate the sample into the various fractions and a syringe or tube may then be interconnected with a portion of the buoy system to extract the selected fractions. For example, a first buoy may be tuned to a density less than the density of a red blood cell fraction of a whole blood sample, bone marrow aspirate sample, or combinations thereof, and a second buoy may be tuned to a density less than the density of a buffy coat fraction.

According to various embodiments, a method of forming an enriched scaffold for application relative to an anatomy is provided. The method may include obtaining a volume of a first whole material and obtaining a volume of a second whole material. A first fraction of the first whole material and a second fraction of the second whole material may be formed. At least one of the first fraction or the second fraction may be applied to the scaffold and at least one fraction for application relative to an anatomy is provided. The method may include obtaining a volume of heterogeneous whole material and separating the material into the desired fraction(s). At least one of the fractions may be applied to the scaffold.

According to various embodiments, a method of withdrawing a material directly from a patient and collecting a selected fraction of the material in a container is provided. The method may include forming an access to port to the patient. A pressure differential in a collection container may be formed relative to the patient. A connection may be made between the patient and the collection container via the port. The collection container may be filled with the material and then the material may be separated to form the selected fraction.

According to various embodiments, a method for concentrating bone aspirate can include obtaining a volume of bone marrow aspirate from a mammal and loading the volume bone marrow aspirate into a separator, the separator operable to separate the aspirate into three of more fractions. The method also includes centrifuging the separator to create a fraction that is a concentrated bone marrow aspirate and extracting and removing the fraction from the separator.

According to various embodiments, a method for concentrating bone marrow aspirate and blood includes collecting bone marrow aspirate and blood from a patient then loading the bone marrow aspirate and blood into a separator that can separate the aspirate and the blood into three or more fractions. The method includes centrifuging the separator containing the bone marrow aspirate and the blood creating a fraction that has a concentrated bone marrow aspirate and a concentrated blood. In various embodiments, such a concentration may be referred to as a buffy coat. The method also include withdrawing the fraction comprising the concentrate or buffy coat.

According to various embodiments, a method for treating a defect in a mammal using a concentrated bone marrow aspirate includes drawing bone marrow aspirate from the mammal and loading the bone marrow aspirate into a separator that can separate the bone marrow aspirate into three or more fractions. The method includes centrifuging the separator separating the bone marrow aspirate into fractions and one fraction is concentrated bone marrow aspirate. The method also can include the concentrated bone marrow aspirate and applying the bone marrow aspirate to a site of a defect in the mammal.

According to various embodiments, a method for treating a defect in a patient includes drawing bone marrow aspirate and whole blood from the patient then adding anticoagulants to the bone marrow aspirate and the blood. The method includes the loading of the bone marrow aspirate and the blood into a separator that can separate the bone marrow aspirate and blood into three or more fractions. The method also includes centrifuging the separator then withdrawing a fraction comprising at least one of the group consisting of hematopoietic stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, red blood cells, white blood cells, fibroblasts, reticulacytes, adipose cells, and endothelial cells, then applying the fraction to the site of the defect in the patient.

According to various embodiments, a method of treating a patient with a combination of concentrated bone marrow aspirate and buffy coat is provided. The method includes obtaining blood and bone marrow aspirate from the patient, forming a buffy coat fraction of the whole blood and forming a concentrated bone marrow aspirate fraction, and applying at least one of the buffy coat or concentrated bone marrow aspirates to the patient.

Further areas of applicability of the present teachings will become apparent from the detailed description provided hereinafter. It should be understood that the detailed description and specific examples, while indicating various embodiment of the teachings, are intended for purposes of illustration only and are not intended to limit the scope of the teachings.

BRIEF DESCRIPTION OF THE DRAWINGS

The present teachings will become more fully understood from the detailed description and the accompanying drawings, wherein:

FIG. 1 is a plan view of a separator including a depth gage affixed to a plunger in a tube according to various embodiments of the present teachings;

FIG. 2 is a cross-section view taken along line 2-2 of FIG. 1;

FIG. 3 is an exploded of the separator apparatus according to various embodiments;

FIG. 4 is a kit including the separator according to an embodiment of the present teachings;

FIG. 5A is a plan view of the separator, according to various embodiments, being filled;

FIG. 5B is a plan view of a blood sample in the separator after the centrifuge process;

FIG. 5C is a plan view of the plunger plunged into the tube with the depth gage to further separate the blood sample;

FIG. 5D is a plan view of the buffy coat and the plasma fractions being extracted from the separator according to various embodiments;

FIG. 6A is a side plan view of a buoy system according to various embodiments;

FIG. 6B is a cross-sectional view of the buoy system of FIG. 6 a;

FIG. 7A is a plan view of a separator, according to various embodiments, being filled;

FIG. 7B is a plan view of a separator, according to various embodiments, after a centrifugation process;

FIG. 7C is a plan view of a separator system, according to various embodiments, being used to extract a selected fraction after the centrifugation process;

FIG. 7D is a plan view of a second fraction being extracted from the separator according to various embodiments;

FIG. 8 is a schematic view illustrating an assisted blood withdrawal device according to various embodiments; and

FIG. 9 is a block diagram illustrating a method for applying selected fractions of a fluid.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

The following description of various embodiments is merely exemplary in nature and is in no way intended to limit the teachings, its application, or uses. Although the following description exemplary refers to a bone reaming material, whole blood and/or bone marrow aspirate separation, it will be understood that the present teachings may be used to separate and concentrate any appropriate material. It will be further understood that many multi-component materials containing particles may be separated. The components or fractions are generally intermingled in the whole sample but may be separated with a centrifuge device that causes increased local gravity or gravitational forces.

With reference to FIGS. 1-3, according to various embodiments, a separator 10, also referred to as a concentrator, is illustrated. The separator 10 generally includes a tube or container 12 that is adapted to hold a fluid sample, such as an anti-coagulated whole blood sample, for further processing. It will be understood that the tube 12 may hold other solutions including constituents of more than one density, such as bone marrow or a mixture of whole blood and bone marrow. The tube 12 includes a top or open end 12 a, which is closeable, and a bottom or closed end 12 b. The bottom 12 b may also be selectively closeable.

Disposed within the tube 12 is a first piston or buoy 14 that is able to move along a central axis A of the tube 12. The buoy 14 is generally nearer the bottom end 12 b of the tube 12 rather than the open end 12 a. The buoy 14 is able to move along a central axis A of tube 12. Also disposed within the tube 12 is a second piston or plunger 16. The plunger 16 is also able to move within the tube 12 generally between a position closer to the open end 12 a to a position closer to the closed end 12 b of the tube 12. A cap 18 substantially mates with the open end 12 a of the tube 12 to close the tube 12 save for ports formed in the cap 18. Extending from the cap 18 is a plasma valve or port 20 that communicates with an area, described further herein, within the tube 12 defined between the plunger 16 and the cap 18. It will be understood that the plasma port 20 is merely exemplary in nature and simply allows for removal of a selected fraction of a sample such as, for example, plasma from whole blood.

The cap 18 also includes a depth gage port 19. Extending from the plunger 16 and through the depth gage port 19 is a first plunger port 22. A depth guide or gage 24 includes a female connector 26 adapted to connect with the first plunger port 22. The depth gage 24 also includes a depth gage housing or cannula 28. The depth gage housing 28 defines a depth gage bore 30. Incorporated in the depth gage housing 28 and extending distal from the end mating with the plunger 16 is a neck 32. The neck 32 includes external neck threads 34. The external neck threads 34 are adapted to engage appropriate internal threads of a mating member.

The mating member may include a compression nut 36 that mates with the external neck threads 34 to lock a depth gage rod 38 in a predetermined position. A split bushing 39 is also provided to substantially seal the depth gage housing 28 when the depth gage rod 38 is locked in place. The depth gage rod 38 extends through the depth gage housing 28 and terminates at a rod handle 40. The rod handle 40 may be a form easily manipulated by a human operator. The depth gage rod 38 extends coaxially with axis A of the tube 12. The depth gage rod 38 extends through the plunger 16 a predetermined distance and may be locked at that distance with the compression nut 36.

Although the tube 12 is described herein as a cylinder, it will be understood that other shapes may be used, such as polygons. The internal portions, such as the cap 18, buoy 14, and plunger 16, would also include this alternate shape. Preferably the tube 12 is formed of a thermal plastic material which is flexible under the forces required to separate blood. The tube 12 may be made of a material that includes the properties of both lipid and alcohol resistance. These properties helps increase the separation speed and decrease the amount of material which may cling to the tube wall 42. For example, Cyrolite MED2® produced by Cyro Industries of Rockaway, N.J., may be used to produce the tube 12.

The tube 12 has a tube wall 42 with a thickness of between about 0.01 millimeters and about 30.0 millimeters, although the tube wall 42 may be any appropriate thickness. The thickness of the tube wall 42 allows the tube wall 42 to flex during the centrifuge process yet be rigid enough for further processing of a blood sample disposed in the tube 12. The tube 12 is closed at the bottom end 12 b with a tube bottom 44 formed of the same material as the tube wall 42 and is formed integrally therewith. Generally, the tube bottom 44 has a thickness which is substantially rigid under the forces required to separate the sample such that it does not flex.

The buoy 14 includes an upper or collection face 46 that defines an inverse cone or concave surface. Generally, the cone has an angle of between about 0.5° to about 45°, and may be about 0.5° to about 90° from a vertical axis, wherein the apex of the cone is within the buoy 14. The collection face 46 forms a depression in the buoy 14 which collects and concentrates material during the separation process. Additionally, the buoy 14 has a bottom face 48 that defines an inverse cone, dome, or covered surface. The buoy bottom face 48 includes an apex 50 that engages the tube bottom 44 before a buoy edge 52 engages the tube bottom 44. The buoy 14 includes a material that is a substantially rigid such that the buoy edges 52 never meet the tube bottom 44. Therefore, there is a gap or free space 54 formed between the buoy edges 52 and the tube bottom 44 along the perimeter of the buoy 14.

The separator 10 is generally provided to separate a multi-component fluid that generally includes various components or constituents of varying densities that are commingled or mixed together. The separator 10 includes the buoy 14 that is of a selected density depending upon a selected constituent of the multi-constituent liquid. Although the buoy 14 may be tuned or of any selected density, the following example relates to separation of whole blood to various components. Therefore, the buoy 14 will be discussed to include a selected density relative to whole blood separation. It will be understood, however, that the buoy 14 may be of any appropriate density depending upon the multi-component fluid being separated including, for example, the concentrating of bone marrow aspirate.

The buoy 14 may be formed of any appropriate material that may have a selected density. For example, when the separator 10 is to separate blood, the buoy 14 generally has a density which is greater than that of red blood cells in a whole blood sample, but less than the plasma or non-red blood cell fraction of a whole blood sample. For blood, the density of the buoy 14 may be between about 1.00 g/cc to about 1.12 g/cc or between about 1.02 g/cc and about 1.09 g/cc.

To achieve the selected density, the buoy 14 may be formed as a composite or multi-piece construction, including a plurality of materials. Particularly, a first or outside portion 56 defines the upper or collection face 46 and the buoy edges 52 and is formed of the same material as the tube 12. The outside portion 56 defines a cup or void into which a plug or insert 58 is placed. The insert 58 has a mass such that the density of the entire buoy 14 is within the selected range, for example, the range described above. Generally, a high density polyethylene may be used, but the material and size of the insert 58 may be altered to produce the desired density of the buoy 14. Alternatively, the buoy 14 may be formed of a single suitable material that has a density in the selected range. Nevertheless, the buoy 14 formed unitarily or of a single material would still include the other portions described in conjunction with the buoy 14.

The outside portion 56 of the buoy 14 also defines the outside circumference of the buoy 14. The outside circumference of the buoy 14 is very close to the internal circumference of the tube 12. Due to the operation of the buoy 14, however, described further herein, there is a slight gap between the outside of the buoy 14 and the inside of the tube 12. Generally, this gap is between about 1 and about 10 thousandths of an inch around the entire circumference of the buoy 14. Generally, it is desired that the distance between the outside circumference of the buoy 14 and the inside circumference of the tube 12 is great enough to allow a selected material or component to pass. For example, in whole blood the distance is selected so that red blood cells may pass through the gap without being lysed, damaged, or activated.

The plunger 16 includes a plunger front or collection face 60 and a plunger wall 62 that extends from the plunger front face 60. The plunger wall 62 extends relatively perpendicular to the plunger front face 60 and substantially parallel to the tube wall 42. Extending from the center of the plunger 16 is a sample collection projection 64. Extending from the top of the sample collection projection 64 is the first plunger port 22. The sample collection projection 64 includes a plunger sample collection bore 68 defined therethrough. The plunger sample collection bore 68 terminates at a sample collection aperture 70 that is substantially in the center of the plunger front face 60. The plunger front face 60 also defines an inverse cone where the sample collection aperture 70 is the apex of the cone. The plunger front face 60 defines a cone with an angle substantially similar or complimentary to the collection face 46 of the buoy 14. In this way, the plunger front face 60 may mate substantially completely with the collection face 46 for reasons described more fully herein.

The plunger 16 also includes a back face 72. Extending from the plunger front face 60 to the back face 72 is a bore 74. A check valve 76 is operably connected to the bore 74. The check valve 76 allows a liquid to move from the plunger front face 60 to the back face 72 while not allowing the liquid to move from the back face 72 to the plunger front face 60. Therefore, the check valve 76 is substantially a one-way valve which allows a material to move in only one direction. The check valve 76 may also operate automatically allowing flow in only one predetermined direction. Alternatively, the check valve 76 may be operated manually and include a portion extending from the check valve 76 requiring manipulation to stop or start a flow through the check valve 76.

The plunger 16 may be made out of any appropriate material which does not interfere with the separation of the fractions of the fluid, such as whole blood. The plunger 16, however, is made of a material that is flexible or at least partially deformable. A flexible material allows the plunger 16 to have an external circumference defined by the plunger walls 62 that is substantially equal to the internal circumference of the tube 12. Because of the deformability of the plunger 16, however, the plunger 16 is still able to move within the tube 12. The plunger 16 is able to move through the tube 12 and also substantially wipe the interior of the tube wall 42. This creates, generally, a moveable seal within the tube 12. Thus, substantially no material escapes the action of the separator 10 when the plunger 16 is plunged into the tube 12. This also helps concentrate the portion of the sample desired to be collected, described more fully herein.

The cap 18 provides a structure to substantially close the tube 12. The cap 18 particularly includes a plate 78 that has an external circumference substantially equal to the external circumference of the tube 12. Extending from the plate 78 and into the tube 12 is a flange 80. The external circumference of the flange 80 is substantially equal to the internal circumference of the tube 12. In this way, the cap 18 substantially closes the tube 12. It will be understood the cap 18 may be in any form so long as the cap 18 substantially closes and/or seals the tube 12 when installed.

Formed through the center of the plate 78 is the depth gage port 19. The depth gage port 19 is also adapted to receive the sample collection projection 64. The first plunger port 22 extends above the plate 78 through the depth gage port 19. The circumference of the depth gage port 19 is substantially equal to the external circumference of the sample collection projection 64 such that a liquid seal is formed. The plate 78 defines a sample face 84 that includes an interior side of the cap 18. The area between the sample face 84 of the cap 18 and the back face 72 of the plunger 16 define a plasma collection area 86. Although the plasma collection area 86 is exemplary called the plasma collection area, it will be understood that the plasma collection area 86 may also collect any appropriate fraction of the sample that is positioned within a separator 10. The plasma collection area 86 is merely an exemplary name and an example of what material may be collected in the area of the separator 10. As discussed herein, the separator 10 may used to separate whole blood into various fractions, therefore, the plasma collection area 86 is used to collect plasma. The plasma collection area 86 also allows a space for the check valve 76 to be installed.

A second bore 88 is formed in the plate 78. Extending through the second bore 88 is the plasma collection valve 20. In liquid communication with the plasma collection valve 20 is a plasma collection tube 92. The plasma collection tube 92 has a length such that the plasma collection tube 92 is able to extend from the plasma collection valve 20 to substantially the tube bottom 44. The plasma collection tube 92, however, is flexible enough such that it may be folded or compressed to fit within the plasma collection area 86 when the plunger 16 is substantially near the open end 12 a of the tube 12. The plasma collection tube 92 may also be connected to a hose barb 93 that includes a plasma collection bore 93 a. The plasma collection bore 93 a is substantially level with the plunger back face 72. Alternatively, the plasma collection bore 93 a may be positioned below the plunger back face 72 but in fluid communication with the plasma collection tube 92.

The outboard side of the plasma collection valve 20 may include external threads 94 to mate with internal threads of a plasma valve cap 96. Therefore, the plasma collection valve 20 may be selectively opened and closed via the plasma valve cap 96. It will be understood, however, that other appropriate means may be used to open and close the plasma collection valve 20 such as a clip or a plug. It will be understood that the plasma collection valve 20, plasma collection tube 92, plasma collection bore 23 a may be used to collect any appropriate material or fraction from the separator 10.

Also formed in the plate 78 is a vent bore 98. The vent bore 98 allows air to flow into the plasma collection area 86 as the plunger 16 is being plunged into the tube 12. The vent bore 98 may include a filter 100 such that liquid cannot escape from the tube 12. The filter 100 allows air to enter or escape from the plasma collection area 86 while maintaining the liquid seal of the tube 12 produced by the cap 18.

Selectively attachable to the first plunger port 22 is the depth gage 24. The female connector 26 interconnects the depth gage housing 28 to the first plunger port 22. Internal threads in the female connector 26 mate with an external thread 102 formed on the first plunger port 22. It will be understood, however, that other engagement mechanisms between the depth gage 24 and the plunger 16 may be used. For example, a snap connection rather than a threaded connection between the two may be used.

The depth gage housing 28 is formed to be substantially rigid. Suitable materials, when sized properly, include polycarbonate and CYRO MED2®. The material preferably is both rigid and does not substantially react with the sample. It is rigid enough to provide a mechanism to plunge the plunger 16 into the tube 12. In addition, the external circumference of the depth gage housing 28 is substantially equal to the circumference of the depth gage port 19 in the plate 78. Therefore, as the plunger 16 is being plunged into the tube 12 with the depth gage 24, no liquid material is allowed to escape around the depth gage housing 28 and through depth gage port 19.

Formed within the depth gage housing 28 is the bore 30 which receives the depth gage rod 38. The depth gage rod 38 extends through the plunger sample collection bore 68 of the sample collection projection 64 and protrudes through the sample collection aperture 70 a predetermined length. The depth gage rod 38 extends through the sample collection aperture 70 a length such that when an end 104 of the depth gage rod 38 meets the buoy 14, the volume defined by the collection face 46 and the plunger front face 60 is between about 5% and about 30% of the total volume of the sample that the tube 12 holds. The projection of the depth gage rod 38 allows for an easily reproducible collection amount and concentration over several trials.

The compression nut 36 locks the depth gage rod 38 in the predetermined position. Nevertheless, once the plunger 16 has been plunged to the desired depth in the tube 12, the compression nut 36 may be loosened so that the depth gage rod 38 may be removed from the plunger 16 and the depth gage housing 28 without moving the plunger 16. A syringe or other appropriate device may then be affixed to the external neck threads 34 of the depth gage 24 to extract the fraction or phase that is between the plunger front face 60 and the collection face 46. As described further herein, the fraction or phase that is left between the plunger front face 60 and the collection face 46 may be the buffy coat of a whole blood sample. Nevertheless, it will be understood that the fraction between the plunger front face 60 and the collection face 46 may be any appropriate fraction of the sample that is disposed in the separator 10.

The separator 10 may be provided alone or in a kit 200, as illustrated in FIG. 4. The kit 200 may be placed in a tray 202 which is covered to provide a clean or sterile environment for the contents of the kit 200. The kit 200 may include at least a first separator 10 and a second separator 10′. A first depth gage 24 and a second depth gage 24′ are also provided, one for each separator 10, 10′. The kit 200 also generally includes a first syringe 204, including a needle, to draw a biological sample, such as blood from a patient. The first syringe 204 may also be used to place the sample in the first separator 10. After centrifuging the sample a second device or syringe 210 may be used to extract a first fraction of the sample, while a third device or syringe 212 may be used to extract a second fraction of the sample. Also, a tourniquet 214 and other medical supplies, such as gauze 216 and tape 218, may be provided to assist the practitioner. It will be understood the elements of the kit 200 are merely exemplary and other appropriate items or elements may be included.

With reference to FIGS. 5A-5D, a method using the separator 10 is illustrated according to various embodiments. The following example relates specifically to the taking and separating of a sample of whole blood from a patient. Nevertheless, it will be understood that another appropriate biological material may be separated and concentrated using the separator 10. For example, bone marrow may be separated and concentrated using the separator 10. The various fractions of the bone marrow are similar to the fractions of whole blood. Generally, the bone marrow includes a fraction that includes substantially dense material and a second phase that is less dense and has other components suspended therein such as, for example, nucleated cells. The bone marrow sample may be positioned in the separator 10, similarly to the whole blood as described herein, and separated in a substantially similar manner as the whole blood. The separator 10 can then be used to remove nucleated cells from the bone marrow sample (which may be referred to as buffy coat), whereas the separator 10, as described herein, is used to remove the buffy coat from the whole blood which includes platelets and other appropriate materials (which may be referred to as platelet rich plasma (PRP)).

A mixture of whole blood and bone marrow may be positioned in the separator 10 for separation and concentration. Similar methods and steps will be used to separate the mixture of whole blood and bone marrow with a main difference being the material that is separated. It will also be understood that various centrifuge times or forces may be altered depending upon the exact material that is being separated with the separator 10. It will also be understood that the separation of whole blood, bone marrow, or a mixture of whole blood and bone marrow are merely exemplary of the materials that may be separated using the separator 10.

According to various embodiments, and with reference to FIGS. 5A-5D and to a whole blood sample, a sample of whole blood taken from a patient is placed in the tube 12 with an anticoagulant using the first syringe 204 or other appropriate delivery method. In particular, the first syringe 204 may be connected to the first plunger port 22. After which the blood sample is provided to the tube 12 via the sample collection bore 68 and sample collection aperture 70. A cap 220 is then placed over the first plunger port 22 to substantially seal the tube 12.

After the whole blood sample is delivered to the tube 12, the separator 10 is placed in a centrifuge. The second separator 10′, substantially identical to the first, is placed opposite the first separator 10 including the sample in a centrifuge. The second separator 10′ may also include a second sample or may include a blank, such as water, so that the centrifuge is balanced. The second separator 10′ balances the centrifuge by both weight and dynamics.

The separator 10 is then spun in the centrifuge in a range between about 1,000 and about 8,000 RPM. This produces a force between about 65 and about 4500 times greater than the force of normal gravity, as generally calculated in the art, on the separator 10 and the blood sample placed in the separator 10. At this force, the more dense material in a whole blood sample is forced toward the bottom end 12 b of the tube 12. The dense material, such as red blood cells or a red blood cell fraction 222, collects on the tube bottom 44. Because the buoy 14 has a density that is less than the red blood cell fraction 222, it is forced in a direction toward the top end 12 a of the tube 12 in the centrifuge. Nevertheless, because the buoy 14 is denser than a plasma fraction 224, the buoy 14 does not reach the top end 12 a of the tube 12.

The forces also affect the tube wall 42. The forces compress the tube 12 linearly along axis A thereby bowing or flexing the tube wall 42. As the tube wall 42 compresses it increases the diameter of the tube 12 making it easier for the buoy 14 to move in the direction of the top 12 a of the tube 12. In addition, the bottom face 48, defining an inverse cone, helps the initial movement of the buoy 14. Because the buoy 14 is not substantially flat along its bottom, it does not form a vacuum interaction with the tube bottom 44. Therefore, the initial movement of the buoy 14 away from the tube bottom 44 is quicker than if the bottom of the buoy 14 was flat.

During the centrifuge process, the red bloods cells of the red blood cell fraction 222 force the buoy 14 in the direction of the top end 12 a of the tube 12 because the buoy 14 is less dense than the red blood cell fraction 222. Although the whole blood sample, including the red blood cells, is loaded above the buoy 14, the red blood cells are able to move between the buoy 14 and the tube wall 42 because the circumference of the buoy 14 is less than the internal circumference of the tube 12. During the centrifuge process, the buoy 14 stops at an interface of a plasma fraction 224 and the red blood cell fraction 222 because of the selected or tuned density of the buoy 14.

With particular reference to FIG. 5B, the centrifuge process has been completed and the buoy 14 has moved to the interface of the red blood cell fraction 222 and plasma fraction 224. After the centrifuge has slowed or stopped, and before or after the tube 12 has been removed from the centrifuge, the tube wall 42 decompresses which helps support the buoy 14 at the interface position. It is also understood that applying an external pressure to the tube 12 via fingers or another apparatus may help stabilize the buoy 14 during the plunging procedure described herein.

On or near collection face 46 is a middle fraction 226, including a small, yet concentrated, amount of red blood cells, white blood cells, platelets, and a substantial portion of a buffy coat of the blood sample. Although the plasma is also present near the collection face 46 at this point, the solid portions of the buffy coat are more compressed against the collection face 46. The position of the buoy 14 also helps in this matter. Because the buoy 14 is a single body it defines the interface of the plasma fraction 224 and the red blood cell fraction 222. Also the density of the buoy 14 assures that it has not passed into the plasma fraction 224. Therefore, the fractions remain separated after the centrifuge process. In addition because the buoy 14 is tuned to the density of the red blood cell fraction 222, it is not affected by variations in the density of the plasma fraction 224 and the buoy's 14 position is always at the interface of the red blood cell fraction 222 and the plasma fraction 224.

With particular reference to FIG. 5C, the depth gage 24 is affixed to the first plunger port 22 of the sample collection projection 64. After connecting the depth gage 24 to the first plunger port 22, the plunger 16 is plunged into the tube 12 by pushing on the depth gage 24. As this is performed the plasma fraction 224, formed and separated above the buoy 14, is able to flow through the check valve 76 into the plasma collection area 86. This displacement of the plasma fraction 224 allows the plunger 16 to be plunged into the tube 12 containing the blood sample.

The plunger 16 is plunged into the tube 12 until the point where the end 104 of the depth gage rod 38 reaches the buoy 14. The volume left in the collection face 46 is the middle fraction 226 and is determined by the depth gage 24. It may be adjusted by selectively determining the amount that the depth gage rod 38 extends below the plunger front face 60. By adjusting the depth gage 24, the concentration of the middle fraction 226 can be adjusted depending upon the desires of the operator.

The plasma fraction 224 is held in the plasma collection area 86 for later withdrawal. Therefore, the use of the plunger 16 and the buoy 14 creates three distinct fractions that may be removed from the tube 12 after only one spin procedure. The fractions include the red blood cell fraction 222, held between the buoy 14 and the tube bottom 44. The middle or buffy coat fraction 226 is held between the plunger 16 and the buoy 14. Finally, the plasma fraction 224 is collected in the plasma collection area 86. In various embodiments, the middle fraction 226 may be a platelet rich plasma (PRP) fraction and the plasma fraction 224 may be a platelet poor plasma (PPP) fraction.

The middle fraction 226 may be extracted from the tube 12 first, without commingling the other fractions, through the sample collection bore 68. With particular reference to FIG. 5D, the depth gage rod 38 may be removed from the depth gage housing 28. This creates a sample collection cannula which includes the depth gage bore 30, the sample collection bore 68, and the sample collection aperture 70. After the depth gage rod 38 has been removed, the second syringe 210 may be affixed to the depth gage housing 28 via the external neck threads 34. The second syringe 210 may be substantially similar to the first syringe 204.

Before attempting to withdraw the middle fraction 226 the separator 10 may be agitated to re-suspend of the platelets and concentrated red blood cells in a portion of the plasma remaining in the collection face 46. This allows for easier and more complete removal of the middle fraction 226 because it is suspended rather than compressed against the collection face 46. A vacuum is then created in the second syringe 210 by pulling back the plunger 16 to draw the middle fraction 226 into the second syringe 210.

As the middle fraction 226 is drawn into the second syringe 210 the plunger 16 moves toward the buoy 14. This action is allowed because of the vent bore 98 formed in the cap 18. Atmospheric air is transferred to the plasma collection area 86 through the vent bore 98 to allow the middle fraction 226 to be removed. This also allows the movement of the plunger 16 toward the buoy 14. This action also allows the plunger 16 to “wipe” the collection face 46. As the plunger front face 60 mates with the collection area 46 the middle fraction 226 is pushed into the sample collection aperture 70. This ensures that substantially the entire middle fraction 226 collected in the collection area 46 is removed into the second syringe 210. It can also increase the repeatability of the collection volumes. In addition, because the second syringe 210 does not protrude out the sample collection aperture 70, it does not interfere with the collection of the middle fraction 226. Once the plunger front face 60 has mated with the collection face 46 there is substantially no volume between the plunger 16 and the buoy 14.

Once the middle fraction 226 is extracted the second syringe 210 is removed from the first plunger port 22. Also the extraction of the middle fraction 226 leaves the plasma fraction 224 and the red blood cell fractions 222 separated in the tube 12. At this point, a third syringe 212 may be affixed to the plasma collection valve 20. The third syringe 212 is connected to the external threads 94 of the plasma collection valve 20 to ensure a liquid tight connection. It will be understood, however, that another connection mechanism such as a snap or compression engagement may be used to connect the third syringe 212 to the plasma collection valve 20.

A vacuum is then created in the third syringe 212 to draw the plasma fraction 224 from the plasma collection area 86 through the plasma collection tube 92. As discussed above, the plasma collection tube 92 is connected to the hose barb 93. Therefore, the plasma flows through the plasma collection bore 93 a through the hose barb 93, and then through the plasma collection tube 92. It will be understood that the plasma collection tube 92 may alternatively simply rest on the plunger back face 72 to collect the plasma fraction 224. In this way, the plasma fraction 224 may be removed from the blood separator 10 without commingling it with the red blood cell fraction 222. After the plasma fraction 224 is removed, the separator 10 may be dismantled to remove the red blood cell fraction 222. Alternatively, the separator 10 may be discarded in an appropriate manner while retaining the red blood cell fraction 222.

The separator 10 allows for the collection of three of a whole blood sample's fractions with only one centrifugation spin. The interaction of the buoy 14 and the plunger 16 allows a collection of at least 40% of the available buffy coat in the whole blood sample after a centrifuge processing time of about 5 minutes to about 15 minutes. The complimentary geometry of the plunger front face 60 and the collection face 46 help increase the collection efficiency. Although only the cone geometry is discussed herein, it will be understood that various other geometries may be used with similar results.

The plunger front face 60 being flexible also helps ensure a complete mating with the collection face 46. This, in turn, helps ensure that substantially the entire volume between the two is evacuated. The process first begins with the suction withdrawal of the middle fraction 226 via the second syringe 210, but is completed with a fluid force action of the middle fraction 226 as the plunger front face 60 mates with the collection face 46. As the plunger front face 60 mates with the collection face 46, the fluid force assists in removal of the selected fraction.

The plunger 16 also substantially wipes the tube wall 42. Because the plunger 16 is formed of a flexible material it forms a seal with the tube wall 42 which is movable. Therefore, substantially no liquid is able to move between the plunger wall 62 and the tube wall 42. Material is substantially only able to go past the plunger front face 60 via the check valve 76.

The complimentary geometry also helps decrease the collection time of the middle fraction 226. Therefore, entire time to prepare and remove the middle fraction 226 is generally about 5 to about 40 minutes. This efficiency is also assisted by the fact that the separator 10 allows for the removal of the middle fraction 226 without first removing the plasma fraction 224, which includes the buffy coat, and re-spinning the plasma fraction 224. Rather, one spin in the separator 10 with the whole blood sample allows for the separation of the buffy coat for easy extraction through the plunger 16.

As discussed above, the separator 10 may be used to separate any appropriate multi-component material. For example, a bone marrow sample may be placed in the separator 10 to be centrifuged and separated using the separator 10. The bone marrow sample may include several fractions or components that are similar to whole blood fractions or may differ therefrom. Therefore, the buoy 14 may be altered to include a selected density that is dependent upon a density of a selected fraction of the bone marrow. The bone marrow may include a selected fraction that has a different density than another fraction and the buoy 14 may be designed to move to an interface between the two fractions to allow for a physical separation thereof. Similar to the whole blood fraction, the plunger 16 may then be moved to near a collection face 46 of the buoy 14. The fraction that is then defined by the collection face 46 and the plunger 16 may be withdrawn, as described for the removal of the buffy coat from the whole blood sample. For example, the middle fraction 226 in the bone marrow sample may include a fraction of undifferentiated or stem cells. In various embodiments, the middle fraction 226 in a bone marrow sample may include hematopoietic, stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, red blood cells, white blood cells, fibroblasts, reticulacytes, adipose cells, or endothelial cells. In various embodiments, the middle fraction 226 is concentrated bone marrow aspirate.

It will also be understood that mixtures of various fluids may be separated in the separator 10. For example, a mixture of whole blood and bone marrow may be positioned in the separator 10 at a single time. The buoy 14 may be tuned to move to an interface that will allow for easy removal of both the buffy coat, from the whole blood sample, and the undifferentiated cells, from the bone marrow sample. Nevertheless, it will be understood that the separator 10 may be used within any appropriate biological material or other material having multiple fractions or components therein. Simply, the buoy 14 may be tuned to the appropriate density and the plunger 16 may be used to cooperate with the buoy 14 to remove a selected fraction.

According to various embodiments and with reference to FIGS. 6A and 6B, a buoy system 300 is illustrated. The buoy system 300 generally includes a first buoy or fraction separator member 302 and a second buoy member or fraction separator 304. The first buoy 302 and the second buoy 304 may be operably interconnected with a buoy system cylinder or member 306. The buoy system 300 may be placed in a tube, such as the tube 12. The tube 12 may be formed of any appropriate material, such as the Cryolite Med® 2 as discussed above. Nevertheless, the buoy system 300 may be designed to fit in the tube 12 or may be formed to fit in any appropriate member that may be disposed within a selected centrifuging device. It will be understood that the following discussion relating to buoy system 300 to be substantially matched to the size of the tube 12 is merely exemplary. As the buoy 14 may be sized to fit in any appropriate tube, the buoy system 300 may also be sized to fit in any appropriate tube. It will be further understood that the tube 12 may be any appropriate shape. The tube 12 need not only be cylindrical but may also be or include conical portions, polygonal portions, or any other appropriate shapes.

The first buoy 302 of the buoy system 300 may be generally similar in geometry to the buoy 14. It will be understood that the first buoy member 302 may be formed in the appropriate manner including shape or size to achieve selected results. Nevertheless, the first buoy member 302 generally includes an exterior diameter that may be slightly smaller than the interior diameter of the tube 12. Therefore, the first buoy member 302 may be able to move within the tube 12 during the centrifugal process. Also, as discussed above, the tube 12 may flex slightly during the centrifuging process, thus allowing the first buoy member 302 to include an exterior diameter substantially equivalent to the interior diameter of the tube 12. As discussed further herein, during the centrifugation process, a portion of the fraction of a sample may pass between the exterior wall of the first buoy member 302 and the tube 12.

The first buoy member 302 may generally include a density that is substantially equivalent to a first or selected fraction of the sample. If the sample to be separated includes whole blood and is desired to separate the red blood cells from the other portions of the sample, the first buoy member 302 may have a selected density that may be about 1.00 grams per cc (g/cc) to about 1.10 g/cc. It will be understood that the density of the first buoy member 302 may be any appropriate density, depending upon the fraction to be separated, and this range of densities is merely exemplary for separating red blood cells from a whole blood sample.

In addition, the first buoy member 302 includes a collection face or area 308 at a proximal or upper portion of the first buoy member 302. The collection face 308 generally defines a concave area of the first buoy member 302 and may have a selected angle of concavity. The buoy assembly 300 defines a central axis D. The collection face 308 defines a surface E that is formed at an angle γ to the central axis D of the buoy system 300. The angle γ may be any appropriate angle and may be about 0.5° to about 90°. The angle γ may, however, be between about 45° and 89.5°. Nevertheless, it will be understood that the angle γ may be any appropriate angle to assist in collection of a selected fraction or portion of the sample by the first buoy member 302.

A bottom or lower surface 310 of the first buoy member 302 may define a bottom face. The bottom face 310 may also be formed at an angle D relative to the central axis D. The bottom surface 310 defines a surface or plane F that may be formed at an angle Δ relative to the central axis D of the buoy system 300. The angle Δ may be any appropriate angle and may be about 90° to about 160°. For example, the angle Δ may be about 15°. Similarly to the buoy bottom face 48, the bottom surface 310 defines an apex 312 that may first engage the bottom 12 d of the tube 12, such that most or the majority of the bottom surface 310 does not engage the tube 12. As illustrated further herein, the apex 312 allows for a free space or gap to be formed between the bottom face 310 of the first buoy member 302 and the bottom 12 b of the tube 12.

The second buoy member 304 may include an outer diameter substantially equivalent to the outer diameter of the first buoy member 302. Therefore, the second buoy 304 may move with the first buoy 302, particularly if the second buoy 304 is interconnected with the first buoy 302 with the buoy central cylinder 306. Nevertheless, the second buoy member 304 may be allowed to move substantially freely within the tube 12 during the centrifuging process.

The second buoy member 304 also includes an upper or superior surface 314 that defines a plane G that is formed at an angle relative to the central axis D of the buoy system 300. The angle c of the plane G relative to the central axis D of the buoy system 300 may be any appropriate angle. For example, the angle ε may be about 90° to about 150°. Generally, the angle ε may assist in allowing a selected fraction or a portion of the sample to pass over the top surface 314 and past the second buoy member 304 during the centrifuging process.

The second buoy member 304 also define a bottom or inferior surface 316 that also defines a plane H that may be formed at an angle K relative to the central axis D of the buoy system 300. The angle K may be any appropriate angle, such as about 90° to about 150°. Nevertheless, the angle K may be substantially complimentary to the angle γ of the collection face 308 of the first buoy member 302. For example, if the angle γ is about 80°, the angle K may be about 100°, such that substantially 180° or a straight line is formed when the first buoy member 302 engages the second buoy member 304. This may be for any appropriate reason, such as extraction of a fraction that may be disposed near the collection face 308 of the first buoy member 302. Nevertheless, the angle K may be any appropriate angle as the angle γ.

The second buoy member 304 may be formed to include any appropriate density. For example, the second buoy member 304 may include a density that is less than the plasma fraction of a whole blood sample. It will be understood that the second buoy member 304 may include any appropriate density and a density that is less than the plasma fraction of a whole blood sample is merely exemplary. Nevertheless, if a whole blood sample is desired to be separated and the plasma sample is to be substantially separated from another fraction, the second buoy member 304 may include a density that is less than the plasma fraction of the whole blood sample. Therefore, the density of the second buoy member 304 may be about 0.01 g/cc to about 1.03 g/cc. As described herein, if the second buoy member 304 includes a density less than the plasma fraction of a whole blood sample and the first buoy member 302 includes a density greater than that of the red blood cells, the buoy system 300 may be substantially positioned near an interface between the red blood cell fraction and the plasma fraction of a whole blood sample. Therefore, as discussed above, and further described herein, the platelet or buffy coat fraction of the whole blood sample may be substantially collected near or in the collection face 308 of the buoy system 300.

The buoy post 306 may operably interconnect the first buoy member 302 and the second buoy member 304. The buoy post 306 may be any appropriate connection member. The buoy post need not be a single cylindrical portion. For example the buoy post 306 may include one or more members interconnecting the first buoy member 302 and the second buoy member 304, such as around a perimeter thereof. In addition, the buoy post 306 may include any appropriate shape or geometry.

The buoy system post 306 may be rigidly affixed to the first buoy member 302 and the second buoy member 304, such that the first buoy member 302 may not move relative to the second buoy member 304 and vice versa. Alternatively, the buoy post 306 may be slide ably connected to either or both the first buoy member 302 and the second buoy member 304. According to various embodiments, the buoy post 306 is generally fixedly connected to the first buoy member 302 and slide ably interconnected to the second buoy member 304. The buoy post 306 may include a catch portion or lip 320 that is able to engage a portion of the second buoy member 304, such that a range of travel of the second buoy member 304, relative to the first buoy member 302 is limited. Nevertheless, the range of travel of the second buoy member 304 toward the first buoy member 302 may be substantially unlimited until the second buoy member 304 engages the first buoy member 302.

In various embodiments, the buoy post 306 may also define a central cannula or bore 322. The post bore 322 may include a connection portion 324 substantially defined near an upper or a proximal end of the buoy post 306. This may allow for interconnection of various components with the buoy post 306, such that various components may be moved through the bore 322 from an exterior location. The buoy post 306 may also define a port or cannula 326 that connects the post cannula 322 with the collection face 308. Therefore, a substance may travel through the post cannula 322 and through the port 326. Various substances may then be provided to or removed from the collection face 308 of the first buoy member 302.

In various embodiments, the buoy system 300 may be used to separate a selected multi component sample, such as a whole blood sample. With continuing reference to FIGS. 6A and 6B, and reference to FIGS. 7A-7D, a method of using the buoy system 300, according to various embodiments, is illustrated and described. With reference to FIGS. 7A-7D, like reference numerals are used to indicate like portions of the tube 12 and the associated mechanisms described in FIGS. 1-3. Therefore, it will be understood that the buoy system 300 may be used with the tube 12 or any other appropriate tube or container system or apparatus. Nevertheless, for simplicity, the description of a method of use of the buoy system 300 will be described in conjunction with the tube 12.

The tube 12 may include the cap 18 that further defines a plasma valve or port 20. Extending through the cap 18 and interconnecting with a first flexible tube or member 92, the plasma port 20 may be used to extract a selected fraction of the sample that is positioned above the second buoy member 304. The tube 12 may also define a second port 21, which may also be referred to as a platelet rich plasma (PRP) port. As discussed herein, a second flexible member, such as a flexible tube 21 a, may interconnect the PRP port 21 and a connection portion 324 of a buoy cylinder 306. As illustrated above, the first tube 92 may also be interconnected with a selected portion of the system, such as the top surface 314 of the second buoy member 304. As illustrated above, a valve may be positioned and is operably interconnect the tube 92 with the upper surface 314 of the second buoy member 304. Nevertheless, such a valve is not necessary and it may be provided merely for convenience.

Other portions of the blood separator system 20, particularly those portions of the tube 12 and the cap 18 that have various valves connected therewith may be included in the tube 12 and used with the buoy system 300. Nevertheless, once the buoy system 300 is interconnected, it may be positioned in the interior of the tube 12 and the syringe 204 used to place a sample into the tube 12. The sample may be expressed from the syringe 204 into the interior of the tube 12 and the sample may be any appropriate sample, such as a whole blood sample. Nevertheless, it will be understood, such as discussed above, various other samples may be used, such as bone marrow samples, a mixture of bone marrow and whole blood or non-biological fluids or materials. It will be understood that two buoys 302 and 304 may generally be near one another when the sample is positioned in the tube 12, but are illustrated apart for clarity of the present discussion.

Also, the sample may be placed in the tube 12 according to various embodiments. As described above, an anticoagulant or other components may be mixed with the whole blood sample, if a whole blood sample is used, before the whole blood sample is positioned within the tube 12. As is apparent to one skilled in the art, an anticoagulant or other components may be mixed with a bone marrow aspirate sample or a sample of bone marrow aspirate and whole blood, if such samples are used before such sample is positioned within the tube 12. The syringe 204 is connected with the plunger port 22 extending from the cap 18, although a plunger may not be used in various embodiments.

After the sample is positioned within the tube 12, as described above, a cap may be positioned over the port 22, such that the sample is not allowed to escape from the tube 12. After the sample is placed in the tube 12 and the cap placed on the port 22, the tube 12 including the sample and the buoy system 300 may be centrifuged.

With reference to FIG. 7B, after a centrifugation of the tube 12, including the buoy system 300, substantially three fractions of the sample may be formed. A first fraction 330 may be positioned between the bottom face 310 and the bottom of the tube 44. A middle fraction 332 may be positioned between the collection face 308 and the bottom surface 316 of the second buoy 304. In addition, a third fraction 334 may be positioned between the upper surface 314 and the cap 18 of the tube 12. Generally, the first fraction 330, the middle fraction 332, and the third fraction 334 are substantially physically separated with the buoy system 300. During the centrifugation process, the tube 12 may flex slightly to allow for ease of movement of the buoy system 300 through the tube 12 and the sample. Nevertheless, the buoy system 300, during the centrifugation process, substantially creates the three fractions 330, 332, and 334 without the operation of an operator. Therefore, the formation of at least three fractions may be substantially simultaneous and automatic using the buoy system 300.

The buoy system 300 substantially separates the fractions 330, 332, and 334, such that they may be easily removed from the tube 12. For example, with reference to FIG. 7C, a syringe or other instrument 340 may be used to extract the middle fraction 332 by interconnecting a cannula or bored tube 342 with the connection portion 324 of the buoy cylinder 306. By drawing the plunger 344 into the extraction syringe 340, a vacuum or upward force is produced within the extraction syringe 340. This force draws the middle fraction 332 through the ports 326 of the buoy post 306 and through the buoy cannula 322. Therefore, the middle fraction 332 may be extracted from the tube 12 without substantially commingling the middle fraction 332 with either the first fraction 330 or the third fraction 334. The middle fraction 332 is drawn in the direction of arrow M through the cannula 322 and into the extraction syringe 340.

It will be understood that the second tube 21 a may also be used. The extraction syringe 340 may be interconnected with the PRP port 21 that is interconnected with the connection portion 324 of the buoy cylinder 306. As discussed herein, the buoy cylinder allows access to the middle fraction 332 (platelet rich portion) between the buoy portions. Thus, it will be understood, that access may be obtained and the middle fraction 332 (platelet rich portion or buffy coat of the sample), between the two buoys, may be extracted in a plurality of ways. The illustrations and method described herein is merely exemplary. For example, if bone marrow aspirate is used as the sample, the PRP port 21 would allow for extraction of undifferentiated nucleated cells. In various embodiments, the PRP port 21 allows for extraction of the buffy coat.

Alternatively, if the post 306 is not provided other portions may be provided to gain access to the middle fraction 332. For example, if a plurality of members are provided around the perimeter of the first buoy 302 and the second buoy 304 a valve portion, such as a puncture-able valve, may be provided in the second buoy 304 to be punctured with an object. In this way an extraction needle may puncture the valve to gain access to the middle fraction 332. Regardless, it will be understood that the buoy system 300 may be able to form a plurality of fractions, such as the three fractions 330, 332, and 334 and at least the middle fraction 332 may be extracted without substantially commingling the various fractions.

During the extraction of the middle fraction 332 through the cannula 322, the second buoy member 304 may move in the direction of arrow M toward the first buoy member 302. As described above, the collection face 308 of the first buoy member may include an angle γ that is substantially complementary to the bottom face 316 of the second buoy member 304. Therefore, if the second buoy member 304 is allowed to move along the buoy cylinder 306, the bottom face 316 of the second buoy member 304 may be able to substantially mate with the collection face 308 of the first buoy member 302. Alternatively, if the second buoy member 304 is not allowed to move, the second buoy member may be provided with a vent port or valve, such that the extraction of the middle fraction 332 from the collection face 308 may not be hindered by the buildup of undesirable forces. Nevertheless, if the second buoy member 304 may move, the interaction of the bottom face 316 of the second buoy member 304 may assist in substantially removing the entire middle fraction 332 from the tube 12. As described above, the bottom face 60 of the plunger 16 may also serve a similar purpose when engaging the collection face 46 of the buoy 14.

With reference to FIG. 7D, once the middle fraction 332 has been extracted from the tube 12, the second buoy member 304 may substantially mate with a portion of the first buoy member 302. As discussed above, the second buoy member 304 may substantially only mate with the first buoy member 302 if the second buoy member 304 is able to substantially move relative to the first buoy member 302. Therefore, it will be understood that the second buoy member 304 need not necessarily mate with the first buoy member 302 and is merely exemplary of an operation of various embodiments. Nevertheless, once the middle fraction 332 has been extracted from the tube 12, the port 20 may be used in conjunction with a selected instrument, such as a plasma extraction syringe 212 to remove the plasma or the third fraction 334 from the tube 12 using the extraction tube 92 interconnected with the port 20.

As described above, the tube 92 allows for extraction of the third fraction 334 from the tube 12 without commingling the third fraction 334 with the remaining first fraction 330 in the tube 12. Therefore, similar to the separator and extraction system 10, three fractions may be substantially formed within the tube 12 with the buoy system 300 and may be extracted without substantially commingling the various fractions. Once the third fraction 334 is extracted from the tube 12, the buoy system 300 may be removed from the tube 12, such that the first fraction 330 may be removed from the tube 12. Alternatively, the first fraction 330 may be discarded with the tube 12 and the buoy system 300 as a disposable system. Alternatively, the system may be substantially reusable, such that it can be sterilized and may be sterilized for various uses.

The description of the method of use of the buoy system 300 is exemplary of a method of using a system according to various other embodiments. It will be understood, however, that various specifics may be used from various embodiments to allow for the extraction of selected fractions. For example, the centrifugation process may be substantially a single step centrifugation process. The buoy system 300, according to various embodiments, may allow for the formation of three fractions during a single centrifugation process. This centrifugation process may occur at any appropriate speed, such as about 1000 RPM to about 8000 RPM. This speed may produce a selected gravity that may be approximately 4500 times greater than the normal force of gravity. Nevertheless, these specifics are not necessary to the operation of the buoy system 300 according to various embodiments. The buoy system 300, according to various embodiments, may be used to extract a plurality of fractions of a sample after only a single centrifuging process and without substantially commingling the various fractions of the sample.

With reference to FIG. 8, the blood collection and separation system that includes the tube 12, according to various embodiments, may be filled with a multi-component fluid or solution, such as blood from a patient, is illustrated. The tube 12 may include any appropriate separation system, such as the separation system 300. Nevertheless, in addition to filling the tube 12 with a fluid from the syringe 204 any appropriate method may be used to fill the tube 12. For example, when a solution, including a plurality of components, is placed into the tube 12 it may be collected directly from a source.

For example, a patient 350 may be provided. The patient 350 may be provided for a selected procedure, such as generally an operative procedure or other procedure that requires an intravenous connection 352, such as a butterfly needle, to be provided in the patient 350. The intravenous connection 352 generally provides a tube 354 extending therefrom. The tube 354 may be used to withdraw fluids from the patient 350 or provide materials to the patient 350, such as medicines or other selected components. Nevertheless, the intravenous connection 352 is generally provided for various procedures and may be used to fill the tube 12.

The tube 354 may interconnect with the plunger port 22 or any appropriate portion of the tube 12. The port 22 may be used to connect with the tube 354 in a similar manner as it would connect with the syringe 204, if the syringe 204 was provided. Nevertheless, it will be understood that the tube 354 may be provided directly to the tube 12 from the patient 350. This may reduce the number of steps required to fill the tube 12 and reduce possible cross-contamination from the patient 350 with the various components. Moreover, making a connection directly with the patient 350 may make the withdrawal and collection of blood from the patient 350 more efficient.

Once the tube 354 is interconnected with the tube 12 the pressure differential between the patient 350, such as the intravenous pressure of the blood, may be used to fill the tube 12 to a selected volume. In addition, a vacuum system 356 may be provided The vacuum system 356 may include a vacuum inducing portion or member 358, such as a resilient bulb. The vacuum inducing member 358 may be interconnected with the tube 12 through a selected connecting portion 360.

The vacuum connecting portion 360 may interconnect with an orifice 362. The orifice 362 may be interconnected or extend from the cap 18 or provided in any appropriate portion with the tube 12. Nevertheless, a first one-way valve 364 may be provided along the connection portion 360 or near the orifice 362. The first one-way valve 364 provides that a flow of a fluid, such as a gas, may pass in a first direction but not in a second. A second one-way valve 366 may also be provided downstream from the first one-way valve 364. In this way, a vacuum may be created with the vacuum inducing member 358, such that air is drawn out of the tube 12 and removed through the second one-way valve 366 in the direction of arrow V. Due to the first and second one-way valves 364, 366 the air is generally withdrawn from the tube 12 without substantially allowing the air to flow back into the tube 12. Thus, a vacuum can be created within the tube 12 to assist with removing a selected volume of fluid, such as blood, from the patient 350.

Because the tube 12 may be filled substantially directly from the patient 350, the collection of the fluid, such as blood, may be provided substantially efficiently to the tube 12. Although any appropriate mechanism may be used to assist in withdrawing the blood from the patient 350 the vacuum system 356 may be provided including the vacuum inducing member 358. Any appropriate vacuum creating device may be used, such as a mechanical pump or the like. Nevertheless, the tube 12 may be filled for use during a selected procedure.

As discussed above, the tube 12 may be used to separate a selected portion of the blood obtained from the patient 350 substantially intraoperatively. Therefore, the collection or separation of the various components may be substantially autologous and substantially intraoperatively. Moreover, obtaining the fluid directly from the patient 350 may increase the efficiency of the procedure and the efficiency of the intraoperative or the operative procedure.

With reference to FIG. 9, the separator 10 may be used to separate any appropriate material. The material may be separated for any purpose, such as a surgical procedure. For example, a selected fraction of a bone marrow aspirate or a bone marrow portion may be produced with the separator 10 according to various embodiments. The selected fraction of the bone marrow aspirate may include various components, such as undifferentiated cells. The various undifferentiated cells may be positioned in a selected scaffold or relative to a selected portion of a patient for providing a volume of the undifferentiated cells to the patient. As known to those skilled in the art, a selected portion of the patient may include bone, cartilage, connective tissue, or any other tissue. Also as known by those skilled in the art, a selected portion of the patient may include a defect in bone, cartilage, connective tissue and/or any other tissue. It will be understood that the method described according to FIG. 9 is merely exemplary of various embodiments that may be used to provide a selected fraction of a bone marrow aspirate or other material to a patient or selected position. The selected portion may be placed on the scaffold in any appropriate manner, such as by spraying, dipping, infiltrating, or any appropriate method.

A method of selecting or creating a selected fraction of a bone marrow aspirate in a selected scaffold according to a method 400 is illustrated in FIG. 9. Generally, the method 400 may start in block 402 in obtaining a bone marrow aspirate volume. The bone marrow aspirate (BMA) may be obtained in any selected or generally known manner. For example, a selected region of bone, such as a portion near an operative procedure, may be used to obtain the bone marrow aspirate. Generally, an accessing device, such as a syringe and needle, may be used to access an intramedullary area of a selected bone. The BMA may then be withdrawn into the syringe for various procedures. Once a selected volume of the BMA is obtained in block 402, the BMA may be positioned in the separator 10 according to various embodiments in block 404. The BMA may be positioned in any appropriate separator, such as those described above including the separator 10. Once the BMA is positioned in the separator 10, a selected fraction of the BMA may be separated from the BMA in block 406.

BMA is a complex tissue comprised of cellular components (that contribute to bone growth) including red and white blood cells, their precursors and a connective tissue network termed the stroma. Bone marrow stromal cells or mesenchymal stem cells have the potential to differentiate into a variety of identifiable cell types including osteoblasts, fibroblasts, endothelial cells, reticulocytes, adipocytes, myoblasts and marrow stroma. The selected fraction of the BMA may include undifferentiated cells or any appropriate portion of the BMA. The fractionation or separation of various fractions of the BMA may allow for a volume of BMA to be taken from a single location and the separation or concentration of the selected portion may be performed in the separator 10. Generally, obtaining a small volume of the selected portion from a plurality of locations may be used to obtain an appropriate volume of BMA or selected fraction of the BMA. Nevertheless, the separator 10 may allow for separating a selected volume from a single location from which the BMA is obtained. This may reduce the time of a procedure and increase the efficiency of obtaining the selected fraction of the BMA.

In addition to obtaining a volume of the BMA in block 402, a volume of whole blood may be obtained in block 408. The volume of blood obtained in block 408, according to any appropriate procedure, including those described above, may then be positioned in the separator 10, in block 410. The whole blood may be positioned in any appropriate separator, such as those described above or a separator to separate a selected fraction of the whole blood. As described above, the whole blood may be separated into an appropriate fraction, such as a fraction including a platelet portion or buffy coat. The whole blood may be separated into selected fractions in block 412. It will be understood that the BMA and the whole blood volume may be obtained substantially simultaneously or consecutively in block 402 and 408. In various embodiments, concentrated BMA, such as the middle fraction 332, 226 comprising nucleated cells in separator 10 has a concentration of nucleated cells that is at least 4 times the concentrate of nucleated cells in BMA. Similarly, the selected fractions of the BMA obtained in block 406 and whole blood obtained in block 412 may also be performed substantially sequentially or simultaneously. For example, the separator 10 including the volume of the BMA may be positioned in a separating device, such as a centrifuge, substantially opposite, so as to balance, the separator 10 including the volume of the whole blood. Therefore, a single separation, such as centrifuge procedure may be used to separate both the BMA and the whole blood into selected fractions. This again may increase the efficiency of the procedure to provide both a selected fraction of the BMA and a selected fraction of the whole blood substantially simultaneously.

The selected fractions of the BMA and the whole blood, provided in block 406 and 412 may be harvested in block 414. The selected fractions of the BMA and the whole blood, may be harvested in block 414 for appropriate purposes, such as those described herein. The separator 10 may be used to obtain the selected fractions of the BMA and the whole blood, through various procedures, such as those described above.

A plurality of separators 10 may be used to obtain a larger quantity of the selection fractions and such quality of selected fractions may be pooled together. In various embodiments, the BMA aspirate can be concentrated alone or in combination with whole blood. In various embodiments, whole blood may be added to the separator 10, and the resulting buffy coat fraction (middle fraction 332, 226) may not only contain the at least 4 times greater concentration of nucleated cells from bone marrow, and may include at least 5 times greater concentration of white blood cells from the whole blood and at least 8 times greater concentration in platelets from the whole blood. In addition, circulating stem cells from whole blood may be concentrated with the mature white blood cells in the buffy coat.

After harvesting the selected fractions of the BMA and the whole blood in block 414, the selected fraction of the BMA may be positioned on an appropriate scaffold in block 416. The scaffold in block 416 may be any appropriate scaffold, such as synthetic bone substitutes or allergenic tissue. Examples of a scaffold include, but are not limited to, bone, cartilage, bone substrates, ceramics, biopolymers, collagens, metal, and the like. The scaffolds may be used for appropriate procedures, such as hard or soft tissue grafting, including uses in non-union or chronic wounds. The undifferentiated cells of the BMA may allow for a substantial source of cells for use during a substantially natural healing after an operative procedure, for example, the natural healing of a patient may use the supplied undifferentiated cells. In such a natural healing of a patient, the undifferentiated cells may be applied to a wound, a defect, a graft site, a bone, cartilage, connective tissue. and the like. Therefore, the scaffold may be positioned in a selected portion of the anatomy and the cells may be allowed to grow and differentiate into selected portions in the implanted position.

It is well known to those skilled in the art that bone marrow contains hematopoietic and mesenchymal stems cells that are precursors for most of the cells found within the body. In general, the hematopoietic cells, along with angiogeneic growth factors, such as those found in platelets, will promote angiogenesis. Angiogenesis is a necessary stage in wound healing of most tissues and treatment of ischemia. The mesenchymal stem cells are the cells responsible for the formation of bone, tendon, ligament, articular cartilage, muscle, fat, intervertebral discs, meniscus, skin, and any other structural tissue found in the body. In various embodiments, a concentration and delivery of these precursor cells, with or without platelet concentrate, will improve therapeutic uses over the native bone marrow.

In addition to positioning the selected fractioning of the BMA and the scaffold in block 416, the platelets of the whole blood may be positioned on or near the scaffold of block 418. The platelets of the whole blood fraction positioned in the scaffold of block 418 may assist the undifferentiated cells and the anatomy into which the scaffold is positioned to allow for a substantially efficient and complete healing. The platelet rich fraction of the whole blood sample may include various healing and growth factors that may assist in providing an efficient and proper healing in the anatomy. Therefore, the undifferentiated cells of the BMA, or other selected fraction obtained from the separation of the BMA, and the selected fraction of the whole blood, obtained from the separator, may be used with the scaffold to provide a substantially efficient implant.

In some embodiments, harvest block 414 may be combined with a growth factors which may include any of the well-known growth factors such as Platelet-Derived Growth Factor (PDGF), Transforming Growth Factor Beta (TGF-β), Insulin-Like Growth Factor (IGF), Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF), Bone Morphogenetic Proteins (BMPs), and vectors for gene therapy. In various embodiments, harvest block 414 may be combined with cellular solutions, suspensions, and materials including osteoblasts, osteoprogenitor cells, chondroblasts, stem cells, or fibroblasts may also be used, as may solutions or suspensions containing other therapeutic agents such as antibiotics, analgesics, pharmaceutical agents, antithrombinolytics, or chemotherapeutic agents. In various embodiments, the buffy coat, platelet fraction, and/or undifferentiated cell fraction (the middle fraction 332, 226) may be combined with an activator such as thrombin solutions and the like. In various embodiments the middle fraction 332, 226 may be used alone for cartilage repair. In various embodiments, the platelet rich fraction may be used to fill a cartilage defect with a fibrin matrix, or it may be mixed with other cell sources such as autologous chondrocytes, synovial cells, bone marrow cells, or to mix with the blood clot formed during microfracture. In addition, the separator 10, or any appropriate separator, such as that described above, may allow for a substantially quick and efficient separation of the BMA and the whole blood into an appropriate fraction for use in the procedure. Other examples include pooling whole blood and/or BMA from different sites of the anatomy or from different sources.

In various embodiments, the concentrated bone marrow cells can also be included with a carrier to aide in delivery and to help maintain the cells' location after implantation. Examples of carriers can include fibrin, concentrated fibrin, demineralized bone matrix, gelatin, collagen, porous calcium based ceramics, porous metal, synthetic fiber matrices, or resorbable matrices. In addition, carriers can be made from other autogeneic, allogenic, and xenogeneic tissues.

After the selected portion of the BMA and the whole blood are positioned on the scaffold in blocks 416 and 418 the scaffold may be implanted in block 420. As described above, the scaffold may be implanted in any appropriate position in the block 420 for various procedures. It will be understood that the scaffold may be implanted for any appropriate procedure and may allow for positioning the selected portion of the BMA, such as undifferentiated cells, and the selected portion of the whole blood, such as platelets, relative to a selected portion of the anatomy. The scaffold may allow for a bone ingrowth, such as allowed with the undifferentiated cells, to assist in healing of a selected portion of the anatomy. In various embodiments, concentrated bone marrow aspirate can be used in articular cartilage repair. The middle fraction 332, 226 can be added to a focal defect or to an osteoarthritic defect. The defects can be in any joint that contains articular cartilage. In various embodiments, the middle fraction 332, 226 can promote the formation of repair tissue. In addition, these cells can be added to a microfracture technique in order to increase the mesenchymal cells present in the defect and increase the amount and quality of repair tissue that forms. In various embodiments, the middle fraction 332, 226 can be delivered in one of the carriers listed above. In various embodiments, for delivery to cartilage, the middle fraction 332, 226 in an autologous fibrin or concentrated fibrin is activated with an activating solution so that it forms a 3-D gel in situ and holds the middle fraction 332, 226 within the cartilage defect. In various embodiments, the concentrated bone marrow can also be used in meniscus repair. The concentrated bone marrow can be used to fill a tear with the meniscus, or in can be used to soak a graft used to replace the meniscus after a full or partial meniscectomy. In various embodiments, concentrated bone marrow aspirate used in combination with platelet rich plasma can also be used for repair of meniscus. In various embodiments, concentrated bone marrow aspirate can be used to repair bone. In various embodiments, the middle fraction 332, 226 can used alone, or mixed with an appropriate carrier such as demineralized bone matrix, calcium based ceramics, fibrin, or concentrated fibrin. The defects in bone could be found in long bones, cranium, sternum, and spine. One specific placement of concentrated bone marrow aspirate would be to deliver the undifferentiated cells to a freeze dried demineralized bone product (such as the Bonus DBM product from Biomet Biologics) under vacuum. In various embodiments, the concentrated bone marrow can infiltrate the graft, and the plasma in the bone marrow may hydrate the bone matrix and create an injectable carrier. The undifferentiated cells may have differentiating growth factors included within the demineralized bone to stimulate cartilage and bone formation. In various embodiments, the concentrated bone marrow can be delivered to the patient with a growth factor that will induce proliferation, chemotaxis, and/or morphogeneis. Examples of growth factors that could be used include PDGF, TGF-b, IGF, VEGF, EGF, CTGF, FGF, and any of the BMPs.

The description of the invention is merely exemplary in nature and, thus, variations that do not depart from the gist of the invention are intended to be within the scope of the invention. Such variations are not to be regarded as a departure from the spirit and scope of the invention. 

1-32. (canceled)
 33. A method for concentrating bone marrow aspirate, the method comprising: obtaining a volume of bone marrow aspirate; loading the volume of bone marrow aspirate into a separator comprising two buoys and an extraction port, the buoys operable to substantially physically separate the aspirate into three or more fractions and the extraction port operable to allow extraction of a fraction between the buoys without substantially commingling the fraction with the other fractions in the separator; centrifuging the separator; wherein the buoys separate the bone marrow aspirate creating the fraction between the buoys comprising a bone marrow aspirate concentrate, wherein the bone marrow aspirate concentrate includes red blood cells and white blood cells; and extracting the fraction between the buoys of the bone marrow aspirate concentrate through the extraction port.
 34. The method according to claim 33 wherein the at least one fraction comprises at least one of the group consisting essentially of hematopoietic stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, fibroblasts, reticulacytes, adipose cells, and endothelial cells.
 35. The method according to claim 33 wherein an amount of nucleated cells in the bone marrow aspirate concentrate is greater than or equal to 4 times an amount of nucleated cells found in the volume of bone marrow aspirate.
 36. The method according to claim 33 further comprising applying the bone marrow aspirate concentrate into a patient.
 37. The method according to claim 64 wherein the combined volume comprises at least one of the group consisting essentially of hematopoietic stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, fibroblasts, reticulacytes, adipose cells, and endothelial cells.
 38. The method according to claim 64 wherein an amount of nucleated cells in the combined volume is greater than or equal to 4 times an amount of nucleated cells in the volume of bone marrow aspirate and the volume of blood.
 39. A method for treating a defect in a mammal using a concentrated bone marrow aspirate, the method comprising: drawing a volume of bone marrow aspirate from the mammal; loading the volume of bone marrow aspirate into a separator comprising two buoys, the separator being operable to separate the aspirate into three or more fractions; centrifuging the separator; separating the volume of bone marrow aspirate into a plurality of fractions including a fraction comprising a concentrated bone marrow aspirate; extracting the fraction comprising a concentrated bone marrow aspirate; and applying the fraction comprising a concentrated bone marrow aspirate to site of the defect.
 40. The method according to claim 39 wherein the fraction comprising a concentrated bone marrow aspirate is essentially buffy coat including a plurality of undifferentiated cells.
 41. The method according to claim 39 wherein the fraction comprises at least one of the group consisting of hematopoietic stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, red blood cells, white blood cells, fibroblasts, reticulacytes, adipose cells, and endothelial cells.
 42. The method according to claim 39 further comprising combining the fraction with a carrier.
 43. The method according to claim 42 wherein the carrier is selected from the group consisting essentially of collagen, hydrogel, a bioabsorbable polymer, a biopolymer, water, buffered solution, fibrin, concentrated fibrin, demineralized bone matrix, gelatin, porous calcium based ceramics, porous metal, synthetic fiber matrices, resorbable matrices, autogeneic tissue, allogenic tissue, xenogeneic tissue, and combinations thereof.
 44. The method according to claim 39 further comprising combining an activating agent with the fraction.
 45. The method according to claim 39 further comprising combining a pharmaceutical agent with the fraction.
 46. A method for treating a defect in an animal, the method comprising: obtaining a volume of bone marrow aspirate and a volume of whole blood from the animal; loading the volume of bone marrow aspirate into a first separator comprising two buoys, the separator being operable to separate the aspirate into three or more fractions; loading the volume of whole blood into a second separator comprising two buoys, the separator being operable to separate the aspirate into three or more fractions; centrifuging the first separator and the second separators, thereby separating the volume of bone marrow aspirate into a plurality of fractions and separating the volume of whole blood into a plurality of fractions; collecting a fraction of the bone marrow aspirate; collecting a fraction of the whole blood; and applying at least one of the fraction of bone marrow aspirate and the fraction of whole blood to site of the defect.
 47. The method according to claim 46 wherein the at least one of the fraction of bone marrow aspirate and the fraction of whole blood comprises at least one of the group consisting of hematopoietic stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, red blood cells, white blood cells, fibroblasts, reticulacytes, adipose cells, and endothelial cells.
 48. The method according to claim 46 further comprising combining the at least one of the fraction of bone marrow aspirate and the fraction of whole blood with a carrier.
 49. The method according to claim 48 wherein the carrier is selected from the group consisting essentially of collagen, hydrogel, a bioabsorbable polymer, a biopolymer, water, buffered solution, fibrin, concentrated fibrin, demineralized bone matrix, gelatin, porous calcium based ceramics, porous metal, synthetic fiber matrices, resorbable matrices, autogeneic tissue, allogenic tissue, xenogeneic tissue, and combinations thereof.
 50. The method according to claim 48 further comprising adding an activating agent to the at least one of the fraction of bone marrow aspirate and the fraction of whole blood.
 51. The method according to claim 48 further comprising adding a pharmaceutical agent to the at least one of the fraction of bone marrow aspirate and the fraction of whole blood.
 52. A method for treating a defect in a patient, the method comprising: drawing a volume of bone marrow aspirate and a volume of whole blood from the patient; adding a first anticoagulant to the volume of bone marrow aspirate; adding a second anticoagulant to the volume of whole blood; loading the volume of bone marrow aspirate and the volume of whole blood into a separator comprising two buoys, the separator being operable to separate the volume of bone marrow aspirate and the volume of whole blood into three or more fractions; centrifuging the separator, separating the volume of bone marrow aspirate and the volume of whole blood into a plurality of fractions; withdrawing a fraction comprising at least one of the group consisting of hematopoietic stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, red blood cells, white blood cells, fibroblasts, reticulacytes, adipose cells, and endothelial cells; and applying the fraction to site of the defect.
 53. The method according to claim 52 wherein the fraction is essentially buffy coat including a plurality of undifferentiated cells.
 54. The method according to claim 52 further comprising combining the fraction with a carrier.
 55. The method according to claim 63 wherein the carrier is selected from the group consisting essentially of collagen, hydrogel, a bioabsorbable polymer, a biopolymer, water, buffered solution, fibrin, concentrated fibrin, demineralized bone matrix, gelatin, porous calcium based ceramics, porous metal, synthetic fiber matrices, resorbable matrices, autogeneic tissue, allogenic tissue, xenogeneic tissue, and combinations thereof.
 56. The method according to claim 52 further comprising combining an activating agent with the fraction.
 57. The method according to claim 52 further comprising combining a pharmaceutical agent with the fraction.
 58. A method of treating a patient with combination of a concentrated bone marrow aspirate and buffy coat, the method comprising: obtaining a volume of a whole blood from the patient; obtaining a volume of a bone marrow aspirate from the patient; forming a buffy coat fraction of the whole blood; forming a concentrated bone marrow aspirate fraction of the bone marrow aspirate; and applying at least one of the buffy coat fraction or the concentrated bone marrow aspirate fraction to the patient.
 59. The method according to claim 58, wherein the forming a first fraction of the first whole material and the forming a second fraction of the second whole material includes: loading the volume of whole blood and the volume of bone marrow aspirate in a container; and applying a force to the container to form at least three fractions.
 60. The method according to claim 58, wherein the forming a first fraction of the first whole material and the forming a second fraction of the second whole material includes: loading the volume of whole blood and the volume of bone marrow aspirate in the container having a separating member, the separating member having a specific gravity substantially dependent upon at least one of the at least three fractions; and centrifuging the container to move the separating member to a selected position relative to the at least one of the volume of whole blood and the volume of bone marrow aspirate to substantially physically separate the at least three fractions.
 61. The method according to claim 58 wherein the at least one of the first fraction or the second fraction comprises at least one of the group consisting of hematopoietic stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, red blood cells, white blood cells, fibroblasts, reticulacytes, adipose cells, and endothelial cells.
 62. The method according to claim 58 further comprising combining the at least one of the first fraction or the second fraction with a carrier.
 63. The method according to claim 62 wherein the carrier is selected from the group consisting essentially of collagen, hydrogel, a bioabsorbable polymer, a biopolymer, water, buffered solution, fibrin, concentrated fibrin, demineralized bone matrix, gelatin, porous calcium based ceramics, porous metal, synthetic fiber matrices, resorbable matrices, autogeneic tissue, allogenic tissue, xenogeneic tissue, and combinations thereof.
 64. A method for preparing a bone marrow aspirate concentrate and a peripheral blood concentrate, the method comprising: collecting a volume of bone marrow aspirate and a volume of peripheral blood from a patient to create a combined volume; loading the combined volume of bone marrow aspirate and blood into a separator comprising two buoys and an extraction port, the buoys being operable to substantially physically separate the combined volume of aspirate and blood into three or more fractions and the extraction port operable to allow extraction of a fraction between the buoys without substantially commingling the fraction with the other fractions in the separator; centrifuging the separator, wherein the buoys separate the combined volume to create the fraction between the buoys comprising a bone marrow aspirate concentrate and a peripheral blood concentrate fraction comprising red blood cells and white blood cells; and withdrawing the fraction between the buoys of bone marrow aspirate concentrate and peripheral blood concentrate through the extraction port.
 65. The method according to claim 64 further comprising applying the combined volume into a patient. 